Chimeric Polypeptides and Uses Thereof

ABSTRACT

The invention provides novel peptides (e.g., linkers) and polypeptide compositions comprising the linkers (e.g., fusion proteins) and methods of using the polypeptide compositions. Peptides (e.g., linkers) are useful as tags and for engineering fusion proteins (e.g., antigen binding molecules, scFv). Polypeptide linkers described herein facilitate flexibility of linked peptides allowing for proper folding, conformation and reduced immunogenicity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/138,331, filed Sep. 21, 2018, which claims priority to U.S.Provisional Application No. 62/562,223, filed Sep. 22, 2017, both ofwhich are incorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 20, 2018, isnamed KPI-005US1 ST25.txt and is 8,625 bytes in size.

TECHNICAL FIELD

The present disclosure is directed to novel peptides and polypeptidecompositions comprising such peptides (e.g., linkers, chimericpolypeptides, antigen biding molecules) and methods of using andpreparing the same.

BACKGROUND

Antigen binding molecules, including antibodies, are used inimmunotherapy and solid phase-based applications such as biosensors,affinity chromatography, and immunoassays. These antibodies and antigenbinding molecules gain their utility by virtue of their ability tospecifically bind their targets.

Fusion proteins may require linker sequences, which are oftenpeptide-based when employed in biotechnological and biotherapeuticapplications, which may serve a range of scientifically-relevantapplications. For example, a linker may be used as a spacer moiety inorder to impart a desired structural and/or functional property to alarger molecule. In another example, a linker may impart little or nostructural or functional properties to a larger molecule, but may beused simply as a distinguishing feature (e.g., a “marker” or “biomarker”or “tag”), uniquely identifying a larger molecule. In still anotherexample, a linker may be used to impart a recognizable feature that mayserve as a binding site for an antibody directed against a largermolecule comprising the peptide sequence.

SUMMARY

The present invention provides, among other things, novel peptidesequences which allow for the proper expression, folding, identificationand activity of a fusion protein. The novel peptides described herein,may be used for connecting domains within a fusion protein (e.g., scFvs)facilitating flexibility of the individual peptide domains. ScFvscomprise the binding domain of most CAR constructs. A scFv comprises IgGvariable light and heavy chains and a flexible peptides (e.g., linker)connecting these two domains. A linker must be long enough to connectthe domains into a single protein construct. Further, it is desirablethat a linker or tag construct not be a potential cause ofimmunogenicity.

Commonly used linkers include repeats ofglycine-glycine-glycine-glycine-serine (G4S) (SEQ ID NO: 32) due totheir intrinsic flexibility and simplicity of side chains, which may beless immunogenic in therapeutic applications. The 18-residue Whitlowlinker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1), was first described by Whitlowet al. in 1993. The peptides described herein, may also be used as apeptide tag. In some embodiments, the fusion protein comprises apolypeptide fused to a peptide (e.g., a linker) described herein. Novelchimeric polypeptides described herein comprising a consensus sequenceBPXXXZ combine desirable attributes (e.g., flexibility and reducedimmunogenicity) suitable for incorporation into fusion proteins usefulfor therapeutic intervention.

In one aspect, the present invention provides a peptide comprising 6-20amino acids and a consensus sequence BPXXXZ, wherein X is a glycine (G)or serine (S), B is a positively charged amino acid and Z is glycine (G)or a negatively charged amino acid. In one embodiment, the presentinvention provides a peptide, wherein the consensus sequence is KPGSGE(SEQ ID NO: 4). In another embodiment, the consensus sequence is GKPGSGE(SEQ ID NO: 5) or GKPGSGG (SEQ ID NO: 6).

In one aspect, the present invention provides a peptide comprising 6-20amino acids and a consensus sequence BPXXXZ, wherein X is a glycine (G)or serine (S), B is a positively charged amino acid and Z is glycine (G)or a negatively charged amino acid, and wherein the peptide is notGSTSGSGKPGSGEGSTKG (SEQ ID NO: 1), GSGKPGSGEG (SEQ ID NO: 13), GKPGSGEG(SEQ ID NO: 14), or SGKPGSGE (SEQ ID NO: 15).

In one aspect, the present invention provides a peptide comprising 8-20amino acids and a consensus sequence XBPXXXZX, wherein each X isindependently a glycine (G) or serine (S), B is a positively chargedamino acid and Z is glycine (G) or a negatively charged amino acid and Pis proline.

In one aspect, the present invention provides a peptide comprising 8-20amino acids and a consensus sequence XBPXXXZX, wherein each X isindependently a glycine (G) or serine (S), B is a positively chargedamino acid and Z is glycine (G) or a negatively charged amino acid and Pis proline, and wherein the peptide is not GSTSGSGKPGSGEGSTKG (SEQ IDNO: 1), GSGKPGSGEG (SEQ ID NO: 13), GKPGSGEG (SEQ ID NO: 14), orSGKPGSGE (SEQ ID NO: 15).

In some embodiments, the present invention provides a peptide comprising8-20 amino acids and a consensus sequence XBPXXXZX, wherein each X isindependently a glycine (G) or serine (S), B is lysine (K) or arginine(R), and Z is glycine (G) or a negatively charged amino acid, and P isproline.

In some embodiments, the present invention provides a peptide comprising8-20 amino acids and a consensus sequence XBPXXXZX, wherein X isindependently a glycine (G) or serine (S), B is lysine (K), and Z isglycine (G) or a negatively charged amino acid, and P is proline.

In some embodiments, the present invention provides a peptide comprising8-20 amino acids and a consensus sequence XBPXXXZX, wherein each X isindependently a glycine (G) or serine (S), B is a positively chargedamino acid, and Z is glycine (G) and P is proline.

In some embodiments, Z is a negatively charged amino acid selected fromglutamic acid (E) or aspartic acid (D). In some embodiments, Z isglutamic acid (E).

In some embodiments, the present invention provides a peptide, whereinthe consensus sequence is GKPGSGE (SEQ ID NO: 5) or GKPGSGG (SEQ ID NO:6). In some embodiments, the consensus sequence is GKPGSGE (SEQ ID NO:5). In some embodiments, the peptide comprises the consensus sequenceGSGKPGSGEGG (SEQ ID NO: 31).

In some embodiments, the peptide comprises one or more repeats of theconsensus sequence. In some embodiments, the repeats are contiguous. Insome embodiments, the peptide repeats are separated by 1-4 amino acids.In some embodiments, the peptide is xxxGKPGSGExxxGKPGSGExxx (SEQ ID NO:3), wherein X is a glycine (G) or serine (S).

In some embodiments, the peptide is not GSTSGSGKPGSGEGSTKG (SEQ ID NO:1), GSGKPGSGEG (SEQ ID NO: 13), GKPGSGEG (SEQ ID NO: 14), or SGKPGSGE(SEQ ID NO: 15). In certain embodiments, the peptide is notGSTSGSGKPGSGEGSTKG (SEQ ID NO: 1). In certain embodiments, the linker isnot GSGKPGSGEG (SEQ ID NO: 13). In certain embodiments, the peptide isnot GKPGSGEG (SEQ ID NO: 14). In certain embodiments, the peptide is notSGKPGSGE (SEQ ID NO: 15).

In some embodiments, the peptide comprises 6-20 amino acids. In someembodiments, the peptide comprises 10-20 amino acids. In someembodiments, the peptide comprises 14-19 amino acids. In someembodiments, the peptide comprises 15-17 amino acids. In someembodiments, the peptide comprises 15-16 amino acids. In someembodiments, the peptide comprises 16 amino acids.

In some embodiments, the peptide comprises an amino acid sequence ofGGGSGKPGSGEGGGS (SEQ ID NO: 7). In some embodiments, the peptidecomprises an amino acid sequence of GGGSGKPGSGEGGGGS (SEQ ID NO: 8). Insome embodiments, the peptide comprises an amino acid sequence ofGGGGSGKPGSGGGGS (SEQ ID NO: 9). In some embodiments, the peptidecomprises an amino acid sequence of GGGGSGKPGSGEGGS (SEQ ID NO: 10). Insome embodiments, the peptide comprises an amino acid sequence ofGGGGSGKPGSGEGGGS (SEQ ID NO: 11). In some embodiments, the peptidecomprises an amino acid sequence of GGGGSGKPGSGEGGGGS (SEQ ID NO: 12).In some embodiments, the peptide comprises an amino acid sequence ofSTSGSGKPGSGEGST (SEQ ID NO: 17). In some embodiments, the peptidecomprises an amino acid sequence of GGGGSGGGGSGGGGSG (SEQ ID NO: 18). Insome embodiments, the peptide comprises an amino acid sequence ofGGGGGSGGGGSGGGGS (SEQ ID NO: 19). In some embodiments, the peptidecomprises an amino acid sequence of GGGGSGGGGSGGGGGS (SEQ ID NO: 20).

In one aspect, the present invention provides a peptide comprising 8-20amino acids and an amino acid sequence at least 80% identical to any oneof GGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8),GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12),STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18),GGGGGSGGGGSGGGGS (SEQ ID NO: 19), or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).

In some embodiments, the peptide comprises an amino acid sequence thatis at least 90% identical to any one of GGGSGKPGSGEGGGS (SEQ ID NO:7),GGGSGKPGSGEGGGGS (SEQ ID NO:8), GGGGSGKPGSGGGGS (SEQ ID NO:9),GGGGSGKPGSGEGGS (SEQ ID NO: 10), GGGGSGKPGSGEGGGS (SEQ ID NO: 11),GGGGSGKPGSGEGGGGS (SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID NO: 17),GGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID NO: 19), orGGGGSGGGGSGGGGGS (SEQ ID NO: 20).

In one aspect, the present invention provides a peptide comprising 8-20amino acids and an amino acid sequence that contains at least six (6)identical amino acids out of ten (10) contiguous amino acids found inany one of GGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO:8), GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12),STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18),GGGGGSGGGGSGGGGS (SEQ ID NO: 19), or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).

In some embodiments, the peptide amino acid sequence contains at leastseven (7), at least eight (8) or at least nine (9) identical amino acidsout of ten (10) contiguous amino acids found in any one ofGGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8),GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12),STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18),GGGGGSGGGGSGGGGS (SEQ ID NO: 19), or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).

In some embodiments, the peptide comprises an amino acid sequence ofGGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID NO: 19), orGGGGSGGGGSGGGGGS (SEQ ID NO: 20).

In one aspect, the present invention provides a fusion proteincomprising a first polypeptide; a second polypeptide; and a peptidelinker as described herein. In some aspects, the fusion protein is anantigen binding molecule. In some embodiments, the antigen bindingmolecule is a scFv. In some embodiments, the first polypeptide is alight chain variable domain and the second polypeptide is a heavy chainvariable domain. In some embodiments, the fusion protein is a chimericantigen receptor.

In one aspect, the present invention provides a polynucleotide encodinga peptide (e.g., linker, tag) as described herein. In some embodiments,the present invention provides a polynucleotide encoding a fusionprotein as described herein.

In one aspect, the present invention provides an expression vectorcomprising a polynucleotide encoding a peptide (e.g., a linker or fusionprotein) as described herein. In some embodiments, the present inventionprovides a recombinant cell comprising a polynucleotide encoding apeptide (e.g., a linker or fusion protein) as described herein. In someembodiments, the recombinant cell comprises an expression vectorcomprising a polynucleotide encoding a peptide (e.g., a linker or fusionprotein) as described herein.

Any aspect or embodiment described herein may be combined with any otheraspect or embodiment as disclosed herein. While the present inventionhas been described in conjunction with the detailed description thereof,the foregoing description is intended to illustrate and not limit thescope of the present invention, which is defined by the scope of theappended claims. Other aspects, advantages, and modifications are withinthe scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, dictionaries,documents, manuscripts and scientific literature cited herein are herebyincorporated by reference.

Other features and advantages of the invention will be apparent from theDrawings and the following Detailed Description, including the Examples,and the claims.

BRIEF DESCRIPTION OF THE DRAWING

The above and further features will be more clearly appreciated from thefollowing detailed description when taken in conjunction with theaccompanying drawings. The drawings however, are for illustrationpurposes only, not for limitation.

FIG. 1 shows an amino acid sequence alignment of exemplary peptide(e.g., linker) sequences.

FIG. 2 shows a bar graph of results of an epitope mapping ELISAexperiment of peptides comprising SEQ ID NOs 1, 21-25, 1 and 26-30,respectively.

FIG. 3 shows a bar graph of the results of antibody binding profiles ofpolypeptide linkers comprising SEQ ID Nos 32, 9-11 and 17, respectively.

FIGS. 4A-4G are a series of plots showing the results of flow cytometryexperiments performed using cells presenting a chimeric antigen receptor(CAR) comprising the peptide KL2 (SEQ ID NO: 10), KL3 (SEQ ID NO: 11),KL4 (SEQ ID NO: 7), KL5 (SEQ ID NO: 12), KL6 (SEQ ID NO: 8), and G452(SEQ ID NO: 18).

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout theSpecification.

As used in this Specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive and covers both “or” and “and.”

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include A and B, A or B, A (alone),and B (alone). Likewise, the term “and/or” as used in a phrase such as“A, B, and/or C” is intended to encompass each of the following aspects:A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B andC; A (alone); B (alone); and C (alone).

The terms “e.g.,” and “i.e.” as used herein, are used merely by way ofexample, without limitation intended, and should not be construed asreferring only those items explicitly enumerated in the specification.

The terms “or more”, “at least”, “more than”, and the like, e.g., “atleast one” are understood to include but not be limited to at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 ormore than the stated value. Also included is any greater number orfraction in between.

Conversely, the term “no more than” includes each value less than thestated value. For example, “no more than 100 nucleotides” includes 100,99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82,81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64,63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46,45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28,27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included is anylesser number or fraction in between.

The terms “plurality”, “at least two”, “two or more”, “at least second”,and the like, are understood to include but not limited to at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more.Also included is any greater number or fraction in between.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps. It is understood thatwherever aspects are described herein with the language “comprising,”otherwise analogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided.

Unless specifically stated or evident from context, as used herein, theterm “about” refers to a value or composition that is within anacceptable error range for the particular value or composition asdetermined by one of ordinary skill in the art, which will depend inpart on how the value or composition is measured or determined, i.e.,the limitations of the measurement system. For example, “about” or“comprising essentially of” may mean within one or more than onestandard deviation per the practice in the art. “About” or “comprisingessentially of” may mean a range of up to 10% (i.e., ±10%). Thus,“about” may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than thestated value. For example, about 5 mg may include any amount between 4.5mg and 5.5 mg. Furthermore, particularly with respect to biologicalsystems or processes, the terms may mean up to an order of magnitude orup to 5-fold of a value. When particular values or compositions areprovided in the instant disclosure, unless otherwise stated, the meaningof “about” or “comprising essentially of” should be assumed to be withinan acceptable error range for that particular value or composition.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to be inclusive of the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one-tenth and one-hundredth of an integer), unlessotherwise indicated.

Units, prefixes, and symbols used herein are provided using theirSystème International de Unites (SI) accepted form. Numeric ranges areinclusive of the numbers defining the range.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, Juo, “TheConcise Dictionary of Biomedicine and Molecular Biology”, 2nd ed.,(2001), CRC Press; “The Dictionary of Cell & Molecular Biology”, 5thed., (2013), Academic Press; and “The Oxford Dictionary Of BiochemistryAnd Molecular Biology”, Cammack et al. eds., 2nd ed, (2006), OxfordUniversity Press, provide those of skill in the art with a generaldictionary for many of the terms used in this disclosure.

“Administering” refers to the physical introduction of an agent to asubject, using any of the various methods and delivery systems known tothose skilled in the art. Exemplary routes of administration for theformulations disclosed herein include intravenous, intramuscular,subcutaneous, intraperitoneal, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular,intra-arterial, intrathecal, intralymphatic, intralesional,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion, as well as in vivo electroporation. In some embodiments, theformulation is administered via a non-parenteral route, e.g., orally.Other non-parenteral routes include a topical, epidermal, or mucosalroute of administration, for example, intranasally, vaginally, rectally,sublingually or topically. Administering may also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

As used herein, an antigen binding molecule, an antibody, or an antigenbinding molecule thereof “cross-competes” with a reference antibody oran antigen binding molecule thereof if the interaction between anantigen and the first binding molecule, an antibody, or an antigenbinding molecule thereof blocks, limits, inhibits, or otherwise reducesthe ability of the reference binding molecule, reference antibody, or anantigen binding molecule thereof to interact with the antigen. Crosscompetition may be complete, e.g., binding of the binding molecule tothe antigen completely blocks the ability of the reference bindingmolecule to bind the antigen, or it may be partial, e.g., binding of thebinding molecule to the antigen reduces the ability of the referencebinding molecule to bind the antigen. In certain embodiments, an antigenbinding molecule that cross-competes with a reference antigen bindingmolecule binds the same or an overlapping epitope as the referenceantigen binding molecule. In other embodiments, the antigen bindingmolecule that cross-competes with a reference antigen binding moleculebinds a different epitope as the reference antigen binding molecule.Numerous types of competitive binding assays may be used to determine ifone antigen binding molecule competes with another, for example: solidphase direct or indirect radioimmunoassay (MA); solid phase direct orindirect enzyme immunoassay (EIA); sandwich competition assay (Stahli etal., 1983, Methods in Enzymology 9:242-253); solid phase directbiotin-avidin EIA (Kirkland et al., 1986, J. Immunol. 137:3614-3619);solid phase direct labeled assay, solid phase direct labeled sandwichassay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, ColdSpring Harbor Press); solid phase direct label MA using 1-125 label(Morel et al., 1988, Molec. Immunol. 25:7-15), solid phase directbiotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552), anddirect labeled MA (Moldenhauer et al., 1990, Scand. J. Immunol.32:77-82).

An “antigen” refers to any molecule that provokes an immune response oris capable of being bound by an antibody or an antigen binding molecule.The immune response may involve either antibody production, or theactivation of specific immunologically-competent cells, or both. Aperson of skill in the art would readily understand that anymacromolecule, including virtually all proteins or peptides, could serveas an antigen. An antigen may be endogenously expressed, i.e. expressedby genomic DNA, or may be recombinantly expressed. An antigen may bespecific to a certain tissue, such as a cancer cell, or it may bebroadly expressed. In addition, fragments of larger molecules may act asantigens. In one embodiment, antigens are tumor antigens.

The term “allogeneic” refers to any material derived from oneindividual, which is then introduced to another individual of the samespecies, e.g., allogeneic T cell transplantation.

The terms “transduction” and “transduced” refer to the process wherebyforeign DNA is introduced into a cell via viral vector (see Jones etal., “Genetics: principles and analysis,” Boston: Jones & Bartlett Publ.(1998)). In some embodiments, the vector is a retroviral vector, a DNAvector, a RNA vector, an adenoviral vector, a baculoviral vector, anEpstein Barr viral vector, a papovaviral vector, a vaccinia viralvector, a herpes simplex viral vector, an adenovirus associated vector,a lentiviral vector, or any combination thereof.

A “cancer” refers to a broad group of various diseases characterized bythe uncontrolled growth of abnormal cells in the body. Unregulated celldivision and growth results in the formation of malignant tumors thatinvade neighboring tissues and may metastasize to distant parts of thebody through the lymphatic system or bloodstream. A “cancer” or “cancertissue” may include a tumor. Examples of cancers that may be treated bythe methods of the present invention include, but are not limited to,cancers of the immune system including lymphoma, leukemia, myeloma, andother leukocyte malignancies. In some embodiments, the methods of thepresent invention may be used to reduce the tumor size of a tumorderived from, for example, bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, cutaneous or intraocular malignant melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, testicular cancer, uterine cancer, carcinoma ofthe fallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagina, carcinoma of the vulva, multiplemyeloma, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primarymediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma(DLBCL), follicular lymphoma (FL), transformed follicular lymphoma,splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancerof the small intestine, cancer of the endocrine system, cancer of thethyroid gland, cancer of the parathyroid gland, cancer of the adrenalgland, sarcoma of soft tissue, cancer of the urethra, cancer of thepenis, chronic or acute leukemia, acute myeloid leukemia, chronicmyeloid leukemia, acute lymphoblastic leukemia (ALL) (including non Tcell ALL), chronic lymphocytic leukemia (CLL), solid tumors ofchildhood, lymphocytic lymphoma, cancer of the bladder, cancer of thekidney or ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinalaxis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos,other B cell malignancies, and combinations of said cancers. In oneparticular embodiment, the cancer is multiple myeloma. The particularcancer may be responsive to chemo- or radiation therapy or the cancermay be refractory. A refractor cancer refers to a cancer that is notamendable to surgical intervention and the cancer is either initiallyunresponsive to chemo- or radiation therapy or the cancer becomesunresponsive over time.

An “anti-tumor effect” as used herein, refers to a biological effectthat may present as a decrease in tumor volume, a decrease in the numberof tumor cells, a decrease in tumor cell proliferation, a decrease inthe number of metastases, an increase in overall or progression-freesurvival, an increase in life expectancy, or amelioration of variousphysiological symptoms associated with the tumor. An anti-tumor effectmay also refer to the prevention of the occurrence of a tumor, e.g., avaccine.

A “cytokine,” as used herein, refers to a non-antibody protein that isreleased by one cell in response to contact with a specific antigen,wherein the cytokine interacts with a second cell to mediate a responsein the second cell. A cytokine may be endogenously expressed by a cellor administered to a subject. Cytokines may be released by immune cells,including macrophages, B cells, T cells, and mast cells to propagate animmune response. Cytokines may induce various responses in the recipientcell. Cytokines may include homeostatic cytokines, chemokines,pro-inflammatory cytokines, effectors, and acute-phase proteins. Forexample, homeostatic cytokines, including interleukin (IL) 7 and IL-15,promote immune cell survival and proliferation, and pro-inflammatorycytokines may promote an inflammatory response. Examples of homeostaticcytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7,IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examplesof pro-inflammatory cytokines include, but are not limited to, IL-1a,IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta,fibroblast growth factor (FGF) 2, granulocyte macrophagecolony-stimulating factor (GM-CSF), soluble intercellular adhesionmolecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1),vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placentalgrowth factor (PLGF). Examples of effectors include, but are not limitedto, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin.Examples of acute phase-proteins include, but are not limited to,C-reactive protein (CRP) and serum amyloid A (SAA).

“Chemokines” are a type of cytokine that mediates cell chemotaxis, ordirectional movement. Examples of chemokines include, but are notlimited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derivedchemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 orCCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a),MIP-10 (MIP-1b), gamma-induced protein 10 (IP-10), and thymus andactivation regulated chemokine (TARC or CCL17).

A “therapeutically effective amount,” “effective dose,” “effectiveamount,” or “therapeutically effective dosage” of a therapeutic agent,e.g., engineered CAR T cells, is any amount that, when used alone or incombination with another therapeutic agent, protects a subject againstthe onset of a disease or promotes disease regression evidenced by adecrease in severity of disease symptoms, an increase in frequency andduration of disease symptom-free periods, or a prevention of impairmentor disability due to the disease affliction. The ability of atherapeutic agent to promote disease regression may be evaluated using avariety of methods known to the skilled practitioner, such as in humansubjects during clinical trials, in animal model systems predictive ofefficacy in humans, or by assaying the activity of the agent in in vitroassays.

The term “lymphocyte” as used herein includes natural killer (NK) cells,T cells, or B cells. NK cells are a type of cytotoxic (cell toxic)lymphocyte that represent a major component of the inherent immunesystem. NK cells reject tumors and cells infected by viruses. It worksthrough the process of apoptosis or programmed cell death. They weretermed “natural killers” because they do not require activation in orderto kill cells. T-cells play a major role in cell-mediated-immunity (noantibody involvement). Its T-cell receptors (TCR) differentiatethemselves from other lymphocyte types. The thymus, a specialized organof the immune system, is primarily responsible for the T cell'smaturation. There are six types of T-cells, namely: Helper T-cells(e.g., CD4+ cells), Cytotoxic T-cells (also known as TC, cytotoxic Tlymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cells or killerT cell), Memory T-cells ((i) stem memory TSCM cells, like naive cells,are CD45RO−, CCR7+, CD45RA+, CD62L+(L-selectin), CD27+, CD28+ andIL-7Rα+, but they also express large amounts of CD95, IL-2R13, CXCR3,and LFA-1, and show numerous functional attributes distinctive of memorycells); (ii) central memory TCM cells express L-selectin and the CCR7,they secrete IL-2, but not IFNγ or IL-4, and (iii) effector memory TEMcells, however, do not express L-selectin or CCR7 but produce effectorcytokines like IFNγ and IL-4), Regulatory T-cells (Tregs, suppressor Tcells, or CD4+CD25+ regulatory T cells), Natural Killer T-cells (NKT)and Gamma Delta T-cells. B-cells, on the other hand, play a principalrole in humoral immunity (with antibody involvement). They makeantibodies and antigens, perform the role of antigen-presenting cells(APCs), and turn into memory B-cells after activation by antigeninteraction. In mammals, immature B-cells are formed in the bone marrow.

The term “genetically engineered”, “engineered”, or “modified” refers toa method of modifying a cell, including, but not limited to, creating adeficiency in a gene by deleting a coding or non-coding region or aportion thereof or by antisense technology, or increasing expression ofa protein introducing a coding region or a portion thereof. In someembodiments, the cell that is modified is a stem cell (e.g.,hematopoietic stem cell (HSC), embryonic stem cell (ES), inducedpluripotent stem (iPS) cell), lymphocyte (e.g., a T cell), which may beobtained either from a patient or a donor. The cell may be modified toexpress an exogenous construct, such as, e.g., a pre-TCR alpha protein,a chimeric antigen receptor (CAR) or a T cell receptor (TCR), which maybe incorporated into the cell's genome.

An “immune response” refers to the action of a cell of the immune system(for example, T lymphocytes, B lymphocytes, natural killer (NK) cells,macrophages, eosinophils, mast cells, dendritic cells and neutrophils)and soluble macromolecules produced by any of these cells or the liver(including Abs, cytokines, and complement) that results in selectivetargeting, binding to, damage to, destruction of, and/or eliminationfrom a vertebrate's body of invading pathogens, cells or tissuesinfected with pathogens, cancerous or other abnormal cells, or, in casesof autoimmunity or pathological inflammation, normal human cells ortissues.

The term “immunotherapy” refers to the treatment of a subject afflictedwith, or at risk of contracting or suffering a recurrence of, a diseaseby a method comprising inducing, enhancing, suppressing, or otherwisemodifying an immune response. Examples of immunotherapy include, but arenot limited to, T cell therapies. T cell therapy may include adoptive Tcell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy,autologous cell therapy, engineered autologous cell therapy (eACT™), andallogeneic T cell transplantation. However, one of skill in the artwould recognize that the conditioning methods disclosed herein wouldenhance the effectiveness of any transplanted T cell therapy. Examplesof T cell therapies are described in U.S. Patent Publication Nos.2014/0154228 and 2002/0006409, U.S. Pat. No. 5,728,388, andInternational Publication No. WO 2008/081035.

The T cells of the immunotherapy may come from any source known in theart. For example, T cells may be differentiated in vitro from ahematopoietic stem cell population; induced pluripotent stem cells(iPS), embryonic stem cells (ES), or T cells may be obtained from asubject. T cells may be obtained from, e.g., peripheral bloodmononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood,thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In addition, the T cells may bederived from one or more T cell lines available in the art. T cells mayalso be obtained from a unit of blood collected from a subject using anynumber of techniques known to the skilled artisan, such as FICOLL™separation and/or apheresis. Additional methods of isolating T cells fora T cell therapy are disclosed in U.S. Patent Publication No.2013/0287748, which is herein incorporated by references in itsentirety.

The term “engineered Autologous Cell Therapy,” which may be abbreviatedas “eACT™,” also known as adoptive cell transfer, is a process by whicha patient's own T cells are collected and subsequently geneticallyaltered to recognize and target one or more antigens expressed on thecell surface of one or more specific tumor cells or malignancies. A“patient” as used herein includes any human who is afflicted with acancer (e.g., a lymphoma or a leukemia). The terms “subject” and“patient” are used interchangeably herein.

As used herein, the term “in vitro cell” refers to any cell, which iscultured ex vivo. In particular, an in vitro cell may include a T cell.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptidecontains at least two amino acids, and no limitation is placed on themaximum number of amino acids that may comprise a protein or peptide'ssequence. As used herein, peptides of the present invention may functionas a linker (e.g., joining two peptides or polypeptides). As usedherein, peptides of the present invention may function as a biomarker ortag. The terms peptide, tag, or linker are used interchangeably.Polypeptides include any peptide or protein comprising two or more aminoacids joined to each other by peptide bonds. As used herein, the termrefers to both short chains, which also commonly are referred to in theart as peptides, oligopeptides and oligomers, for example, and to longerchains, which generally are referred to in the art as proteins, of whichthere are many types. “Polypeptides” include, for example, biologicallyactive fragments, substantially homologous polypeptides, oligopeptides,homodimers, heterodimers, variants of polypeptides, modifiedpolypeptides, derivatives, analogs, fusion proteins, among others. Thepolypeptides include natural peptides, recombinant peptides, syntheticpeptides, or a combination thereof.

“Stimulation,” as used herein, refers to a primary response induced bybinding of a stimulatory molecule with its cognate ligand, wherein thebinding mediates a signal transduction event. A “stimulatory molecule”is a molecule on a T cell, e.g., the T cell receptor (TCR)/CD3 complex,which specifically binds with a cognate stimulatory ligand present on anantigen present cell. A “stimulatory ligand” is a ligand that whenpresent on an antigen presenting cell (e.g., an APC, a dendritic cell, aB-cell, and the like) may specifically bind with a stimulatory moleculeon a T cell, thereby mediating a primary response by the T cell,including, but not limited to, activation, initiation of an immuneresponse, proliferation, and the like. Stimulatory ligands include, butare not limited to, an anti-CD3 antibody, an MHC Class I molecule loadedwith a peptide, a superagonist anti-CD2 antibody, and a superagonistanti-CD28 antibody.

A “costimulatory signal,” as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to aT cell response, such as, but not limited to, proliferation and/orupregulation or down regulation of key molecules.

A “costimulatory ligand” as used herein, includes a molecule on anantigen presenting cell that specifically binds a cognate co-stimulatorymolecule on a T cell. Binding of the costimulatory ligand provides asignal that mediates a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like. Acostimulatory ligand induces a signal that is in addition to the primarysignal provided by a stimulatory molecule, for instance, by binding of aT cell receptor (TCR)/CD3 complex with a major histocompatibilitycomplex (MEW) molecule loaded with peptide. A co-stimulatory ligand mayinclude, but is not limited to, 3/TR6, 4-1BB ligand, agonist or antibodythat binds Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand,CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), humanleukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), ligand that specifically binds with B7-H3, lymphotoxinbeta receptor, MEW class I chain-related protein A (MICA), MEW class Ichain-related protein B (MICB), OX40 ligand, PD-L2, or programmed death(PD) L1. A co-stimulatory ligand includes, without limitation, anantibody that specifically binds with a co-stimulatory molecule presenton a T cell, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28,CD30, CD40, CD7, ICOS, ligand that specifically binds with CD83,lymphocyte function-associated antigen-1 (LFA-1), natural killer cellreceptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamilymember 14 (TNFSF14 or LIGHT).

A “costimulatory molecule” is a cognate binding partner on a T cell thatspecifically binds with a costimulatory ligand, thereby mediating acostimulatory response by the T cell, such as, but not limited to,proliferation. Costimulatory molecules include, but are not limited to,A “costimulatory molecule” is a cognate binding partner on a T cell thatspecifically binds with a costimulatory ligand, thereby mediating acostimulatory response by the T cell, such as, but not limited to,proliferation. Costimulatory molecules include, but are not limited to,4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19,CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha;beta; delta; epsilon; gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a,CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86,CD8alpha, CD8beta, CD9, CD96 (Tactile), CD1-1a, CD1-1b, CD1-1c, CD1-1d,CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS,GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2Rbeta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD,ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1,LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14;TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1(LFA-1 (CD11a/CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44,NKp46, NKp80 (KLRF1), OX40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD162),signaling lymphocytic activation molecule, SLAM (SLAMF1; CD150; IPO-3),SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr,TNFR2, Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or fragments,truncations, or combinations thereof.

The terms “reducing” and “decreasing” are used interchangeably herein,and indicate any change that is less than the original. “Reducing” and“decreasing” are relative terms, requiring a comparison between pre- andpost-measurements “Reducing” and “decreasing” include completedepletions.

“Treatment” or “treating” of a subject refers to any type ofintervention or process performed on, or the administration of an activeagent to, the subject with the objective of reversing, alleviating,ameliorating, inhibiting, slowing down or preventing the onset,progression, development, severity, or recurrence of a symptom,complication or condition, or biochemical indicia associated with adisease. In one embodiment, “treatment” or “treating” includes a partialremission. In another embodiment, “treatment” or “treating” includes acomplete remission.

To calculate percent identity, the sequences being compared aretypically aligned in a way that gives the largest match between thesequences. One example of a computer program that may be used todetermine percent identity is the GCG program package, which includesGAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics ComputerGroup, University of Wisconsin, Madison, Wis.). The computer algorithmGAP is used to align the two polypeptides or polynucleotides for whichthe percent sequence identity is to be determined. The sequences arealigned for optimal matching of their respective amino acid ornucleotide (the “matched span,” as determined by the algorithm.) Incertain embodiments, a standard comparison matrix (see, Dayhoff et al.,1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A.89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by thealgorithm.

Various aspects of the invention are described in further detail in thefollowing subsections.

DETAILED DESCRIPTION

Compositions are described herein that provide a means to make (e.g.,design, engineer) chimeric or fusion polypeptides. A peptide (e.g.,linker) sequence as described herein allows for the proper expression,folding and activity of a fusion protein. The present inventionprovides, among other things, novel polypeptides (e.g., linkers) andfusion proteins comprising the same. In some embodiments, the presentinvention provides polynucleotide compositions encoding a peptide (e.g.,linker, tag) or fusion protein described herein. In some embodiments,the present invention provides an expression vector comprising thepolynucleotide encoding a peptide (e.g., linker, tag) or fusion protein.In other embodiments, the present invention provides a cell comprisingthe polynucleotide and/or the expression vector encoding a peptide(e.g., linker, tag) or fusion protein. Described herein are novelcompositions comprising peptide linkers, polypeptide compositionscomprising polypeptides joined by the peptide linkers and relatedpolynucleotides, vectors, cells and pharmaceutical compositions. In someembodiments, the peptide (e.g., linker, tag) is fused to one or morepolypeptides. Described linker sequences operably join twopeptides/polypeptides of interest such that the expression and activity(e.g., antigen binding) of the polypeptides connected by the linkers aredurable and optimal. The peptide linker or tag may be fused at theC-terminus, N-terminus, or anywhere within the polypeptide to achievethe desired function.

Peptide Linkers

Novel chimeric polypeptide linkers described herein comprising aconsensus sequence XYPXXXZX combine desirable attributes suitable forincorporation into fusion proteins useful for therapeutic intervention.In one aspect, the present invention provides a linker comprising 8-20amino acids and a consensus sequence XYPXXXZX, wherein X is a glycine(G) or serine (S), B is a positively charged amino acid and Z is glycine(G) or a negatively charged amino acid. The inventors have discoveredthat both the spacing and charge of the of the amino acid residues inthe consensus sequence contribute to functionality of the linker inaddition to antibody recognition of the linker sequence.

In one aspect, the present invention provides a linker comprising 6-20amino acids and a consensus sequence BPXXXZ, wherein X is a glycine (G)or serine (S), B is lysine (K) or arginine (R), and Z is glycine (G) ora negatively charged amino acid, and P is proline.

In some embodiments, the present invention provides a linker comprising8-20 amino acids and a consensus sequence XBPXXXZX, wherein X is aglycine (G) or serine (S), B is lysine (K) or arginine (R), and Z isglycine (G) or a negatively charged amino acid, and P is proline.

In some embodiments, the present invention provides a linker comprising8-20 amino acids and a consensus sequence XBPXXXZX, wherein X is aglycine (G) or serine (S), B is lysine (K), and Z is glycine (G) or anegatively charged amino acid, and P is proline.

In some embodiments, the present invention provides a linker comprising8-20 amino acids and a consensus sequence XBPXXXZX, wherein X is aGlycine (G) or serine (S), B is a positively charged amino acid, and Zis glycine (G), and P is proline.

In some embodiments, Z is a negatively charged amino acid selected fromglutamic acid (E) or aspartic acid (D). In some embodiments, Z isglutamic acid (E).

In some embodiments, the present invention provides a linker, whereinthe consensus sequence is GKPGSGE (SEQ ID NO: 5) or GKPGSGG (SEQ ID NO:6). In some embodiments, the consensus sequence is GKPGSGE (SEQ ID NO:5).

In some embodiments, the peptide comprises an amino acid sequence ofGGGGSGGGGSGGGGSG (SEQ ID NO: 18).

The linker peptide sequence may be of any appropriate length to connectone or more proteins of interest and is preferably designed to besufficiently flexible so as to allow the proper folding and/or functionand/or activity of one or both of the peptides it connects. Thus, thelinker peptide may have a length of no more than 10, no more than 11, nomore than 12, no more than 13, no more than 14, no more than 15, no morethan 16, no more than 17, no more than 18, no more than 19, or no morethan 20 amino acids. In some embodiments, the linker peptide may have alength of at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, or at least 20 amino acids. In some embodiments, the linkercomprises at least 7 and no more than 20 amino acids, at least 7 and nomore than 19 amino acids, at least 7 and no more than 18 amino acids, atleast 7 and no more than 17 amino acids, at least 7 and no more than 16amino acids, at least 7 and no more 15 amino acids, at least 7 and nomore than 14 amino acids, at least 7 and no more than 13 amino acids, atleast 7 and no more than 12 amino acids or at least 7 and no more than11 amino acids. In certain embodiments, the linker comprises 15-17 aminoacids, and in particular embodiments, comprises 16 amino acids. In someembodiments, the linker comprises 10-20 amino acids. In someembodiments, the linker comprises 14-19 amino acids. In someembodiments, the linker comprises 15-17 amino acids. In someembodiments, the linker comprises 15-16 amino acids. In someembodiments, the linker comprises 16 amino acids. In some embodiments,the linker comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 aminoacids.

As used herein, a “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having side chainshave been defined in the art. These families include amino acids withbasic or positively charged side chains (e.g., lysine, arginine,histidine), acidic or negatively charged side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). In certainembodiments, one or more amino acid residues within a polypeptidelinker, fusion protein, CDR(s) or within a framework region(s) of anantibody or antigen binding molecule provided herein (or fragmentthereof) may be replaced with an amino acid residue with a similar sidechain.

Conservative amino acid substitutions, which are encompassed by thepresent disclosure, may encompass non-naturally occurring amino acidresidues, which are typically incorporated by chemical peptide synthesisrather than by synthesis in biological systems. These includepeptidomimetics and other reversed or inverted forms of amino acidmoieties. Naturally occurring residues may be divided into classes basedon common side chain properties:

-   -   hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;    -   neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   acidic (negatively charged): Asp, Glu;    -   basic (negatively charged): His, Lys, Arg;    -   residues that influence chain orientation: Gly, Pro; and    -   aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced, for example, into regions of a humanantibody that are homologous with non-human antibodies, or into thenon-homologous regions of the molecule. Exemplary conservative aminoacid substitutions are set forth in Table A below.

TABLE A Original Residues Exemplary Substitutions Ala Val, Leu, Ile ArgLys, Gln, Asn Asn Gln Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, AlaHis Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala, Phe, Norleucine LeuNorleucine, Ile, Val, Met, Ala, Phe Lys Arg, 1,4 Diamino-butyric acid,Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr,Ala, Cys Thr Ser Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu,Phe, Ala, Norleucine

In some embodiments, the linker comprises an amino acid sequence ofGGGSGKPGSGEGGGS (SEQ ID NO: 7). In some embodiments, the linkercomprises an amino acid sequence of GGGSGKPGSGEGGGGS (SEQ ID NO: 8). Insome embodiments, the linker comprises an amino acid sequence ofGGGGSGKPGSGGGGS (SEQ ID NO: 9). In some embodiments, the linkercomprises an amino acid sequence of GGGGSGKPGSGEGGS (SEQ ID NO: 10). Insome embodiments, the linker comprises an amino acid sequence ofGGGGSGKPGSGEGGGS (SEQ ID NO: 11). In some embodiments, the linkercomprises an amino acid sequence of GGGGSGKPGSGEGGGGS (SEQ ID NO: 12).In some embodiments, the linker comprises an amino acid sequence ofSTSGSGKPGSGEGST (SEQ ID NO: 17). In some embodiments, the peptidecomprises an amino acid sequence of GGGGSGGGGSGGGGSG (SEQ ID NO: 18). Insome embodiments, the peptide comprises an amino acid sequence ofGGGGGSGGGGSGGGGS (SEQ ID NO: 19). In some embodiments, the peptidecomprises an amino acid sequence of GGGGSGGGGSGGGGGS (SEQ ID NO: 20).

In one aspect, the present invention provides a linker comprising 6-20amino acids and an amino acid sequence at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identical to any one of GGGSGKPGSGEGGGS (SEQ ID NO: 7),GGGSGKPGSGEGGGGS (SEQ ID NO:8), GGGGSGKPGSGGGGS (SEQ ID NO: 9),GGGGSGKPGSGEGGS (SEQ ID NO: 10), GGGGSGKPGSGEGGGS (SEQ ID NO: 11),GGGGSGKPGSGEGGGGS (SEQ ID NO: 12), STSGSGKPGSGEGST (SEQ ID NO: 17),GGGGSGGGGSGGGGSG (SEQ ID NO: 18), GGGGGSGGGGSGGGGS (SEQ ID NO: 19) orGGGGSGGGGSGGGGGS (SEQ ID NO: 20).

In some embodiments, the linker amino acid sequence contains at leastfive (5), six (6), seven (7), eight (8) or at least nine (9) identicalamino acids out of ten (10) contiguous amino acids found in any one ofGGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8),GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12),STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18), orGGGGGSGGGGSGGGGS (SEQ ID NO: 19) or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).

In one aspect, the present invention provides a linker comprising 8-20amino acids and an amino acid sequence that contains at least six (6)identical amino acids out of ten (10) contiguous amino acids found inany one of GGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO:8), GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12),STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18),GGGGGSGGGGSGGGGS (SEQ ID NO: 19) or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).

In some embodiments, the linker amino acid sequence contains at leastseven (7), at least eight (8) or at least nine (9) identical amino acidsout of ten (10) contiguous amino acids found in any one ofGGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8),GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),GGGGSGKPGSGEGGGS (SEQ ID NO: 11), or GGGGSGKPGSGEGGGGS (SEQ ID NO: 12),STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18),GGGGGSGGGGSGGGGS (SEQ ID NO: 19) or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).

Fusion Protein

In one aspect, the present invention provides a fusion proteincomprising a first polypeptide; a second polypeptide; and a linker asdescribed herein. Polypeptide composition and polynucleotides encodingthe polypeptide compositions are described herein, in which thepolypeptide compositions comprise a first and secondpeptide/polypeptide, connected by a linker sequence disclosed herein.The inventors have surprisingly found that a linker according to thepresent invention provides both optimal flexibility of the first andsecond peptide and length to avoid steric hindrance and allow correctfolding.

Polypeptide compositions produced in this manner are commonly referredto a fusion or chimeric protein/polypeptides and typically are made bythe expression (e.g., transcription, translation) of nucleic acidsequences encoding the polypeptide compositions, in the appropriatesystem. Means by which to make fusion and/or chimeric polypeptides arewell-known in the art (see for example, Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Springs Harbor Laboratory, 1992) NewYork which is incorporated by reference herein in its entirety).

In the polypeptide compositions described herein, the two polypeptides(e.g., a first polypeptide and a second polypeptide) may berecombinantly joined by any of the linker polypeptides described above,with the linker disposed between the two polypeptides. For example, incertain embodiments, the polypeptides or compositions comprise a firstand a second polypeptide recombinantly joined by a linker comprisingGGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8),GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12),STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18),GGGGGSGGGGSGGGGS (SEQ ID NO: 19) or GGGGSGGGGSGGGGGS (SEQ ID NO: 20).The two polypeptides may be any amino acid sequences includingfull-length proteins, protein fragments or portions, functional proteinfragments or portions, functional protein domains and the like, ofeither two different proteins or the same protein.

As used herein, the term “polypeptide” or “peptide” refers a polymer ofamino acid residues typically joined exclusively by peptide bonds, thatmay be produced naturally (e.g., isolated, essentially purified orpurified) or synthetically (e.g., by chemical synthesis). A polypeptideproduced by expression of a non-host DNA molecule is a “heterologous”peptide or polypeptide. An “amino acid residue” comprising thepolypeptide may be a natural or non-natural amino acid residue linked bypeptide bonds and/or bonds different from peptide bonds. The amino acidresidues may be in D-configuration or L-configuration. In some aspects,the polypeptides referred to herein are proteins, peptides or fragmentsthereof produced by the expression of recombinant nucleic acid. In someembodiments, the polypeptide compositions described herein comprise twopolypeptides connected by a linker sequence.

As used herein, “functional fragment” or “portion” is intended to referto less than the entire mature or native protein which is sufficient toretain one or more of the desired biological activities of the mature ornative protein (e.g., sufficient to retain a therapeutic or ameliorativebiological activity with respect to a disorder to be treated). Thus,amino acid sequences or polypeptides may be modified, for example,polypeptide sequences into which amino acids have been inserted, deletedand/or substituted in such a manner that the modifications do notsubstantially interfere with the polypeptide's ability to encode afunctional agent.

The linker or polypeptide linker described herein refers to a peptidesequence designed to connect (e.g., join, link) two protein sequences,wherein the linker peptide sequence is typically not disposed betweenthe two protein sequences in nature. In the context of the presentinvention, the phrase “linked” or “joined” or “connected” generallyrefers to a functional linkage between two contiguous or adjacent aminoacid sequences to produce a polypeptide that generally does not exist innature. In certain embodiments, linkage may be used to refer to acovalent linkage of, for example, the amino acid sequences of a firstpolypeptide and the second polypeptide (e.g., antibody heavy chain andlight chain). Generally, linked proteins are contiguous or adjacent toone another and retain their respective operability and function whenjoined. Peptides comprising the chimeric polypeptides disclosed hereinare linked by means of an interposed peptide linker comprising one ormore amino acids. Such linkers may provide desirable flexibility topermit the desired expression, activity and/or conformationalpositioning of the chimeric polypeptide. A typical amino acid linker isgenerally designed to be flexible or to interpose a structure, such asan alpha-helix, between the two protein moieties. A linker may be fusedto the N-terminus or C-terminus of a polypeptide, or insertedinternally.

In a polypeptide composition comprising a linker, the 5′ end (e.g.,terminus) of the linker peptide sequence (e.g., amino acid sequence) isadjacent to and covalently linked to the 3′ end of one protein sequence(first peptide) (e.g., full-length protein or protein domain, fragmentor variant) and, further, the 3′ end of the linker amino acid sequenceis adjacent to and covalently linked to the 5′ end of another proteinsequence (second peptide).

Antigen Binding Molecules

In some aspects, the fusion protein is an antigen binding molecule. Insome embodiments, the first polypeptide is a light chain variable domainand the second polypeptide is a heavy chain variable domain. In someembodiments, the use of a linker as described herein to join an antibodyheavy chain and light chain variable region, provides the benefit ofpermitting optimal flexibility and length to avoid steric hindrance andallow correct folding of the antigen binding domains. Properconformation of the first and second peptides is essential for antigenrecognition and binding.

As used herein, the terms “variable region” or “variable domain” areused interchangeably and mean a portion of an antibody, generally, aportion of a light or heavy chain, typically the amino-terminal end ofthe antibody, and comprising about 100-130 amino acids in the heavychain and about 90 to 115 amino acids in the light chain, which differextensively in sequence among antibodies and are used in the binding andspecificity of a particular antibody for a particular antigen. Thevariability in sequence is concentrated in those regions calledcomplementarity determining regions (CDRs) while the more highlyconserved regions in the variable domain are called framework regions(FR). The CDRs of the light and heavy chains are primarily responsiblefor the interaction and specificity of the antibody with antigen.

In certain embodiments, the variable region of an antigen bindingmolecule is a human variable region. In further embodiments, thevariable region comprises rodent, human or murine CDRs and humanframework regions (FRs). In further embodiments, the variable region isa primate (e.g., a non-human primate) variable region. In yet furtherembodiments, the variable region is a rabbit variable region. In otherembodiments, the variable region comprises human CDRs and non-human(e.g., rabbit, murine, rat or non-human primate) framework regions(FRs). In other embodiments, the variable region comprises non-human(e.g., rabbit, murine, rat or non-human primate) CDRs and humanframework regions (FRs).

The terms “VH,” “VH domain” and “VH chain” are used interchangeably andmean the heavy chain variable region of an antigen binding molecule,antibody or an antigen binding fragment thereof. As used herein, theterm “heavy chain” when used in reference to an antibody may refer toany distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ)and mu (μ), based on the amino acid sequence of the constant domain,which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies,respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3 andIgG4.

The terms “VL,” “VL domain” and “VL chain” are used interchangeably andmean the light chain variable region of an antigen binding molecule,antibody or an antigen binding fragment thereof. As used herein, theterm “light chain” when used in reference to an antibody may refer toany distinct type, e.g., kappa (κ) or lambda (λ) based on the amino acidsequence of the constant domains. Light chain amino acid sequences arewell known in the art. In specific embodiments, the light chain is ahuman light chain.

An “antigen binding molecule,” “antigen binding portion,” or “antibodyfragment” refers to any molecule that comprises the antigen bindingparts (e.g., CDRs) of the antibody from which the molecule is derived.An antigen binding molecule may include the antigenic complementaritydetermining regions (CDRs). Examples of antibody fragments include, butare not limited to, Fab, Fab′, F(ab′)₂, and Fv fragments, dAb, linearantibodies, scFv antibodies, and multispecific antibodies formed fromantigen binding molecules. Peptibodies (i.e., Fc fusion moleculescomprising peptide binding domains) are another example of suitableantigen binding molecules. In some embodiments, the antigen bindingmolecule binds to an antigen on a tumor cell. In some embodiments, theantigen binding molecule binds to an antigen on a cell involved in ahyperproliferative disease or to a viral or bacterial antigen. Infurther embodiments, the antigen binding molecule is an antibody orfragment thereof, including one or more of the complementaritydetermining regions (CDRs) thereof. In further embodiments, the antigenbinding molecule is a single chain variable fragment (scFv).

As used herein, the terms “single-chain antibody” and “single chainfragment variable (scFv)” are used interchangeably and mean an antigenbinding molecule in which a VL and a VH region are joined via a linkerto form a continuous protein chain wherein the linker is long enough toallow the protein chain to fold back on itself and form a monovalentantigen binding site (see, e.g., Bird et al., (1988) Science 242:423-26and Huston et al., (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-83(1988). FMC63 (Nicholson et al., (1997) Mol. Immunol. 34: (16-17)1157-65) is a specific example of a scFv, and is specific for CD19.

In some embodiments, the antigen binding molecule is a scFv.

In some embodiments, the present invention provides antigen bindingmolecules, including scFv, that comprise a consensus sequences BPXXXZ,XBPXXXZX or exemplary linker sequence as described herein (e.g., KPGSGE(SEQ ID NO: 4), GKPGSGE (SEQ ID NO: 5), GKPGSGG (SEQ ID NO: 6),GGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8),GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12),STSGSGKPGSGEGST (SEQ ID NO: 17), GGGGSGGGGSGGGGSG (SEQ ID NO: 18),GGGGGSGGGGSGGGGS (SEQ ID NO: 19) or GGGGSGGGGSGGGGGS (SEQ ID NO: 20). Insome embodiments, the molecules comprising these sequences and cellspresenting such molecules, polynucleotides encoding the antigen bindingmolecules are also provided, and form an aspect of the instantdisclosure.

As used herein, the term “binding affinity” means the strength of thesum total of non-covalent interactions between a single binding site ofa molecule (e.g., an antigen binding molecule such as an antibody) andits binding partner (e.g., an antigen). Unless indicated otherwise, asused herein, “binding affinity” refers to intrinsic binding affinitywhich reflects a 1:1 interaction between members of a binding pair(e.g., antibody and antigen). The affinity of a molecule X for itspartner Y may generally be represented by the dissociation constant(Kd). Affinity may be measured and/or expressed in a number of waysknown in the art, including, but not limited to, equilibriumdissociation constant (Kd), and equilibrium association constant (Ka).The Kd is calculated from the quotient of koff/kon, whereas Ka iscalculated from the quotient of kon/koff. kon refers to the associationrate constant of, e.g., an antibody to an antigen, and koff refers tothe dissociation of, e.g., an antibody to an antigen. The kon and koffmay be determined by standard techniques known to one of ordinary skillin the art, such as BIAcore® or KinExA or surface plasmon resonance.

In certain embodiments, an antigen binding molecule comprises a singlechain, wherein the heavy chain variable region and the light chainvariable region are connected by a linker as described herein, to form ascFv (e.g., an antigen binding molecule of instant disclosure). In someembodiments, the VH is located at the N terminus of the linker and theVL is located at the C terminus of the linker. In other embodiments, theVL is located at the N terminus of the linker and the VH is located atthe C terminus of the linker. In some embodiments, the linker comprisesat least about 5, at least about 8, at least about 9, at least about 10,at least about 11, at least about 12, at least about 13, at least about14, at least about 15, at least about 16, at least about 17, at leastabout 18, at least about 19, at least about 20, at least about 25, atleast about 30, at least about 35, at least about 40, at least about 45,at least about 50, at least about 60, at least about 70, at least about80, at least about 90, or at least about 100 amino acids. In someembodiments, the linker comprises between about 8 amino acids and about18 amino acids (e.g., 16 amino acids).

Chimeric Antigen Receptors

An antigen binding molecule may form a component of a CAR or TCR, andmay serve to direct the CAR or TCR to recognize a target of interest. Asused herein, in the context of a CAR or TCR, an antigen binding moleculemeans any component of a CAR or TCR that directs the CAR or TCR to adesired target and associates with that target. In specific embodiments,an antigen binding molecule component of a CAR or TCR comprises a scFvcomprising a heavy and light chain variable region joined by a linkerdescribed herein. The heavy and light variable regions may be derivedfrom the same antibody or two different antibodies. Antigen bindingmolecules used in a CAR or TCR may be derived from an antibody known orsuspect to bind to a target of interest.

T cells may be engineered to express, for example, a chimeric antigenreceptor (CAR) or a T cell receptor (TCR). CAR positive (CAR+) T cellsare engineered to express a CAR. CARs may comprise, e.g., anextracellular single chain variable fragment (scFv) with specificity fora particular tumor antigen, which is directly or indirectly linked to anintracellular signaling part comprising at least one costimulatorydomain, which is directly or indirectly linked to at least oneactivating domain; the components may be arranged in any order. Thecostimulatory domain may be derived from, e.g., CD28 or 4-1BB, and theactivating domain may be derived from, e.g., any form of CD3-zeta. Incertain embodiments, the CAR is designed to have two, three, four, ormore costimulatory domains. A CAR scFv may be designed to target, forexample, CD19, which is a transmembrane protein expressed by cells inthe B cell lineage, including all normal B cells, and B cell malignancessuch as NHL, CLL, and non-T cell ALL. In some embodiments, a CAR isengineered such that the costimulatory domain is expressed as a separatepolypeptide chain. Examples of CAR T cell therapies and constructs aredescribed in U.S. Patent Publication Nos. 2013/0287748, 2014/0227237,2014/0099309, and 2014/0050708, which are incorporated by reference intheir entirety for any purpose.

An antigen binding molecule of the instant disclosure may also be afully human monoclonal antibody, from which a scFv may be generated,which may then form a component of a CAR or TCR provided herein. Fullyhuman monoclonal antibodies may be generated by any number of techniqueswith which those having ordinary skill in the art will be familiar. Suchmethods include, but are not limited to, Epstein Barr Virus (EBV)transformation of human peripheral blood cells (e.g., containing Blymphocytes), in vitro immunization of human B-cells, fusion of spleencells from immunized transgenic mice carrying inserted humanimmunoglobulin genes, isolation from human immunoglobulin V region phagelibraries, or other procedures as known in the art and based on thedisclosure herein.

Procedures have been developed for generating human monoclonalantibodies in non-human animals. For example, mice in which one or moreendogenous immunoglobulin genes have been inactivated by various meanshave been prepared. Human immunoglobulin genes have been introduced intothe mice to replace the inactivated mouse genes. In this technique,elements of the human heavy and light chain locus are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci(see also Bruggemann et al., (1997) Curr. Opin. Biotechnol. 8:455-58).

It will further be appreciated that where desired, the various domainsand regions described herein may be expressed in a separate chain fromthe antigen binding molecule (e.g., scFv) and activating domains, inso-called “trans” configuration. Thus, in one embodiment an activatingdomain may be expressed on one chain, while the antigen bindingmolecule, and/or an extracellular domain, and/or a transmembrane domainand/or a costimulatory domain (depending on the desired construction ofthe CAR or TCR) may be expressed on a separate chain.

Additionally, the N to C-terminal, or extracellular to intracellular,order of the components of a CAR of the instant disclosure may be variedas desired. The antigen binding molecule (the scFv) will beextracellular in order to be associated with the target antigen, and mayinclude a leader or signal peptide at the N terminal end of the scFvthat is most distal to the cell membrane.

Polynucleotides

In one aspect, the present invention provides a polynucleotide encodinga linker as described herein. In some embodiments, the present inventionprovides a polynucleotide encoding a fusion protein as described herein.The instant disclosure is also directed to polynucleotides encodingantibodies and antigen binding molecules, such as a scFv, thatcomprising a linker as described herein, molecules comprising thissequence and cells presenting such molecules.

Expression Vectors

In one aspect, the present invention provides an expression vectorcomprising a polynucleotide encoding a linker or fusion protein asdescribed herein. In certain aspects, provided herein are vectorscomprising a polynucleotide of the instant disclosure. In someembodiments, the instant disclosure is directed to a vector or a set ofvectors comprising a polynucleotide encoding a linker, or fusionprotein, as described herein. In other embodiments, the instantdisclosure is directed to a vector or a set of vectors comprising apolynucleotide encoding an antibody or an antigen binding moleculethereof, as disclosed herein.

Any vector known in the art may be suitable for the instant disclosure.In some embodiments, the vector is a viral vector. In some embodiments,the vector is a retroviral vector, a DNA vector, a murine leukemia virusvector, an SFG vector, a plasmid, a RNA vector, an adenoviral vector, abaculoviral vector, an Epstein Barr viral vector, a papovaviral vector,a vaccinia viral vector, a herpes simplex viral vector, an adenovirusassociated vector (AAV), a lentiviral vector, or any combinationthereof. In some embodiments of the instant disclosure one, two or morevectors may be employed.

Recombinant Cells

In some embodiments, the present invention provides a recombinant cellcomprising a polynucleotide encoding a linker or fusion protein asdescribed herein. In some embodiments, the recombinant cell comprises anexpression vector comprising a polynucleotide encoding a linker orfusion protein as described herein. In some aspects, provided herein arecells comprising a polynucleotide or a vector of the instant disclosure.In some embodiments, the instant disclosure is directed to host cells,such as in vitro cells, comprising a polynucleotide encoding a linker orfusion protein, as described herein. In some embodiments, the instantdisclosure is directed to host cells, e.g., in vitro cells, comprising apolynucleotide encoding an antibody or an antigen binding moleculethereof, as disclosed herein.

Suitable host cells may be derived from a variety of organisms,including, but not limited to, mammals, plants, birds (e.g., aviansystems), insects, yeast, and bacteria. In some embodiments, host cellsare mammalian cells. Any mammalian cell susceptible to cell culture, andto expression of polypeptides, may be utilized in accordance with thepresent invention as a host cell. Non-limiting examples of mammaliancells that may be used in accordance with the present invention includehuman embryonic kidney 293 cells (HEK293), HeLa cells; BALB/c mousemyeloma line (NSO/l, ECACC No: 85110503); human retinoblasts (PER.C6(CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human fibrosarcomacell line (e.g.,HT-1080); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells +/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinomacells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; a human hepatoma line(Hep G2), human cell line CAP and AGE1.HN, and Glycotope's panel.

Non-limiting examples of host cells suitable for the present inventioninclude cells and cell lines derived from Pichia pastoris, Pichiamethanolica, Pichia angusta, Schizosacccharomyces pombe, Saccharomycescerevisiae, and Yarrowia lipolytica for yeast; Sodoptera frugiperda,Trichoplusis ni, Drosophila melangoster and Manduca sexta for insects;and Escherichia coli, Salmonella typhimurium, Bacillus subtilis,Bacillus lichenifonnis, Bacteroides fragilis, Clostridia perfringens,Clostridia difficile for bacteria; and Xenopus Laevis from amphibian.

Additionally, any number of available hybridoma cell lines may beutilized in accordance with the present invention. One skilled in theart will appreciate that hybridoma cell lines might have differentnutrition requirements and/or might require different culture conditionsfor optimal growth and polypeptide or protein expression, and will beable to modify conditions as needed.

EXAMPLES

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same.

Example 1: Epitope Mapping to Identify, Linker Consensus Sequence

The specific binding of antibody Clone 8 and 16 raised against a CARcomprising the linker sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1),were used for epitope mapping ELISA experiments of the full length SEQID NO: 1 and variants truncated on either the N- or C-terminus andcontaining either a biotin moiety on the N-terminus, or a lysine residuewith a biotin moiety on the C-terminus (SEQ ID NOs: 21-30).

The antibodies were captured in 96-well plate format using platespre-coated with Protein G (Pierce). The plates were washed 6× in PBSTbuffer followed by incubation with target peptides. An additional 6×wash was performed with PBST and the antibodies were further incubatedwith streptavidin-HRP. Upon a final 6× wash in PBST, signal was detectedand quantified via enhanced chemiluminescense kit (ECL, from GEHealthcare) and a Varioskan Flash plate reader (Thermo Fisher). Theresults of epitope mapping ELISA experiments, shown in FIG. 2demonstrate that although both antibodies bind to the full length 18mer(SEQ ID NO: 1), Clone 8 specifically binds to the 7mer subsequenceGKPGSGE (SEQ ID NO: 5) and Clone 16 specifically binds to the 5mersubsequence KPGSG (SEQ ID NO: 16). Taken together, these data were usedto generate a consensus sequence based on the minimal binding epitopesfor clone 8 and 16.

Example 2: Antibody Binding Profile of Exemplary Linker Sequences

The specific binding of a panel of antibodies raised against a CARcomprising the linker sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1), thelinker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 2), or the or the anti-CD19scFv clone FMC63 were used to determine the antibody binding profile ofexemplary linker sequences from the KIP-1, KIP-4, and KIP-3 antibodiesrespectively, as described herein. Also included in this assay werepeptides comprising linker sequences KL1 (SEQ ID NO: 9), KL2 (SEQ ID NO:10), KL3 (SEQ ID NO: 11), and the truncated Whitlow linker (SEQ ID NO:17) described in FIG. 1. The antibodies were captured in 96-well plateformat using plates pre-coated with Protein G (Pierce). The plates werewashed 6× in PBST buffer followed by incubation with target peptides. Anadditional 6× wash was performed with PBST and the antibodies werefurther incubated with streptavidin-HRP. Upon a final 6× wash in PBST,signal was detected and quantified via enhanced chemiluminescense kit(ECL, from GE Healthcare) and a Varioskan Flash plate reader (ThermoFisher). The results of the antibody profile ELISA experiments, shown inFIG. 3 demonstrate the breadth of antibody binding of linkers accordingto the present invention.

Example 3: Flow Cytometry Results of CAR Expressing Cells ComprisingChimeric Linkers

CAR T cells were assayed via flow cytometry using Protein L as a controlto confirm the expression of each CAR construct comprising the linkersequences SEQ ID NO: 1 (FMC63 WT), or the SEQ ID NO: 2 (FMC63 G45).These results confirm expression of the CAR constructs on the surface ofT cells. As shown in FIGS. 4A-4G, CAR T cells were produced in thecontext of scFv FMC63 and 24C1 scFv. KL2 (SEQ ID NO: 10), KL3 (SEQ IDNO: 11), KL4 (SEQ ID NO: 7), KL5 (SEQ ID NO: 12), KL6 (SEQ ID NO: 8),and G452 (SEQ ID NO: 18) linkers were used to link the VL and VH domainsof the scFv.

SEQUENCES AND SEQ ID NOS

The instant disclosure comprises a number of nucleic acid andpolypeptide sequences. For convenience, Table B below correlates eachsequence with its appropriate SEQ ID NO.

TABLE B SEQ ID NO Sequence SEQ ID NO: 1 GSTSGSGKPGSGEGSTKG SEQ ID NO: 2GGGGSGGGGSGGGGS SEQ ID NO: 3 xxxGKPGSGExxxGKPGSGExxx SEQ ID NO: 4 KPGSGESEQ ID NO: 5 GKPGSGE SEQ ID NO: 6 GKPGSGG SEQ ID NO: 7 GGGSGKPGSGEGGGSSEQ ID NO: 8 GGGSGKPGSGEGGGGS SEQ ID NO: 9 GGGGSGKPGSGGGGS SEQ ID NO: 10GGGGSGKPGSGEGGS SEQ ID NO: 11 GGGGSGKPGSGEGGGS SEQ ID NO: 12GGGGSGKPGSGEGGGGS SEQ ID NO: 13 GSGKPGSGEG SEQ ID NO: 14 GKPGSGEGSEQ ID NO: 15 SGKPGSGE SEQ ID NO: 16 KPGSG SEQ ID NO: 17 STSGSGKPGSGEGSTSEQ ID NO: 18 GGGGSGGGGSGGGGSG SEQ ID NO: 19 GGGGGSGGGGSGGGGSSEQ ID NO: 20 GGGGSGGGGSGGGGGS SEQ ID NO: 21 GSTSGSGKPGSGEGSTSEQ ID NO: 22 GSTSGSGKPGSGEG SEQ ID NO: 23 GSTSGSGKPGSGE SEQ ID NO: 24GSTSGSGKPGSG SEQ ID NO: 25 GSTSGSGKPG SEQ ID NO: 26 GSGKPGSGEGSTKGSEQ ID NO: 27 SGKPGSGEGSTKG SEQ ID NO: 28 GKPGSGEGSTKG SEQ ID NO: 29KPGSGEGSTKG SEQ ID NO: 30 PGSGEGSTKG SEQ ID NO: 31 GSGKPGSGEGGSEQ ID NO: 32 GGGGS

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) may be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention may be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications, websites and other reference materials referenced hereinto describe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference.

1-14. (canceled)
 15. A fusion protein comprising: a first polypeptideand a second polypeptide covalently linked by a peptide comprising aconsensus sequence GKPGSGZ, wherein the peptide is 6-20 amino acids inlength; wherein Z is glycine (G) or a negatively charged amino acid; andwherein the peptide is not GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1), GSGKPGSGEG(SEQ ID NO: 13), GKPGSGEG (SEQ ID NO: 14), or SGKPGSGE (SEQ ID NO: 15).16. The fusion protein of claim 15, wherein the first polypeptidecomprises a light chain variable domain and the second polypeptidecomprises a heavy chain variable domain.
 17. A polynucleotide encodingthe peptide according to claim
 15. 18. A polynucleotide encoding thefusion protein of claim
 15. 19. An expression vector comprising apolynucleotide of claim
 18. 20. A recombinant cell comprising apolynucleotide of claim
 18. 21. The fusion protein of claim 15, whereinthe consensus sequence is GKPGSGG (SEQ ID NO: 6) or GSGKPGSGEGG (SEQ IDNO: 31).
 22. The fusion protein of claim 15, wherein the peptide is anamino acid sequence having at least 90% identity to any one ofGGGSGKPGSGEGGGS (SEQ ID NO: 7), GGGSGKPGSGEGGGGS (SEQ ID NO: 8),GGGGSGKPGSGGGGS (SEQ ID NO: 9), GGGGSGKPGSGEGGS (SEQ ID NO: 10),GGGGSGKPGSGEGGGS (SEQ ID NO: 11), GGGGSGKPGSGEGGGGS (SEQ ID NO: 12),STSGSGKPGSGEGST (SEQ ID NO: 17),
 23. The fusion protein of claim 15,wherein the fusion protein is an antigen binding molecule.
 24. Therecombinant cell of claim 20, wherein the cell is an immune cell. 25.The immune cell of claim 24, wherein the immune cell is a T cell, NKcell, or a stem cell.